T-cell selective interleukin-4 agonists

ABSTRACT

The invention is directed to human IL-4 muteins numbered in accordance with wild-type IL-4 having T cell activating activity, but having reduced endothelial cell activating activity. In particular, the invention is related to human IL-4 muteins wherein the surface-exposed residues of the D helix of the wild-type IL-4 are mutated whereby the resulting mutein causes T cell proliferation, and causes reduced IL-6 secretion from HUVECs, relative to wild-type IL-4. This invention realizes a less toxic IL-4 mutant that allows greater therapeutic use of this interleukin. Further, the invention is directed to IL-4 muteins having single, double and triple mutations represented by the designators R121A, R121D, R121E, R121F, R121H, R1211, R121K, R121N, R121P, R121T, R121W; Y124A, Y124Q, Y124R, Y124S, Y124T; Y124A/S125A, T13D/R121E; and R121T/E122F/Y124Q, when numbered in accordance with wild type IL-4 (His=1). The invention also includes polynucleotides coding for the muteins of the invention, vectors containing the polynucleotides, transformed host cells, pharmaceutical compositions comprising the muteins, and therapeutic methods of treatment.

This application claims benefit of U.S. Provisional application Ser.Nos. 60/019,748 filed Jun. 14, 1996 and 60/036,746 filed Jan. 27, 1997.

BACKGROUND

1. Field of the Invention

The invention is generally related to the fields of pharmacology andimmunology. More specifically, the invention is directed to novelcompositions of matter for selectively activating T cells, and havingreduced activation of Endothelial cells or fibroblasts. The novelcompositions include variants of the cytokine family, and in particularhuman Interleukin-4 (IL-4).

2. Description of Related Art

Interleukin 4 (IL-4) is a pleiotropic cytokine, having activities oncells of the immune system, endothelium, and those of fibroblasticnature. Reported in vitro effects of IL-4 administration includeproliferation of B cells, immunoglobulin class switching in B cells. InT cells, IL-4 stimulates T cell proliferation after preactivation withmitogens and down-regulates IFN-γ production. In monocytes, IL-4 inducesclass II MHC molecules expression, release of lipopolysaccharide-inducedtPA, and CD23 expression. In Endothelial cells (EC), IL4 inducesexpression of VCAM-1 and IL-6 release, and decreases ICAM-1 expression.(Maher, D W, et al., Human Interleukin-4: An Immunomodulator withPotential Therapeutic Applications, Progress in Growth Factor Research,3:43-56 (1991)).

Because of its ability to stimulate proliferation of T cells activatedby exposure to IL-2, IL-4 therapy has been pursued. For instance, IL-4has demonstrated anti-neoplastic activity in animal models of renalcarcinomas, and has induced tumor regression in mice (Bosco, M, et al.,Low Doses of IL-4 Injected Perilymphatically in Tumor-bearing MiceInhibit the Growth of Poorly and Apparently Nonimmunogenic Tumors andInduce a Tumor Specific Immune Memory, J. Immunol., 145:3136-43 (1990)).However, its toxicity limits dosage in humans (Margolin, K, et al.,Phase II Studies of Human Recombinant Interleukin-4 in Advanced RenalCancer and Malignant Melanoma, J. Immunotherapy 15:147-153 (1994)).

Because of its immunoregulatory activity, a number of clinicalapplications are suggested for IL-4. Among these clinical applicationsare disorders caused by imbalances of the immune system, particularlythose caused by imbalances of T helper (Th) cell responses to antigen.These diseases include certain autoimmune diseases, rheumatic diseases,dermatological diseases, and infectious diseases. A large body ofexperimental work has established that Th cells fall into two broadclasses, designated Th1 and Th2 (Mosmann, T. R., Cherwinski, H., Bond,M. W., Giedlin, M A. and Coffinan, R. L., Two types of murine helper Tcell clone. I. Definition according to profiles of lymphokine activitiesand secreted proteins, J. Immunol., 136:2348-2357 (1986); Mosmann, T.R., Cytokines, differentiation and functions of subsets of CD4 and CD8 Tcells, Behring Inst. Mitt., 1-6 (1995)). These T cell classes aredefined by the cytokines they express: Th1 cells make IL-2, INF-γ, andTNF-α, while Th2 cells make IL-4 and IL-5. Th1 and Th2 cells are formedfrom naive CD4+T cells. Differentiation into Th1 or Th2 subsets dependson the cytokine present during antigen stimulation: IFN-γ and IL-12direct differentiation of naive cells to the Th1 phenotype, while IL-4directs differentiation to the Th2 phenotype. While the Th1 and Th2subsets may represent extremes along a continuum of Th cell phenotypes(for example, Th0 cells, which express low levels of both INF-γ andIL-4, have been described), this classification nevertheless is themajor paradigm in the field of immunology for describing the characterof the immune response.

It has been observed that certain organ-specific autoimmune diseases areassociated with a predominantly Th1 T cell response against autoantigen(Liblau R S; Singer S M, McDevitt H O, Th1 and Th2 CD4+T cells in thepathogenesis of organ-specific autoimmune diseases, Immunol. Today,16:34-38 (1995)). One such autoimmune disease is insulin-dependentdiabetes (IDDM), a disorder characterized by T cell-mediated destructionof pancreatic D cells. Several lines of evidence suggest that Th1 -typecells are primarily responsible for the pancreatic 0 cell destruction(reviewed in Tisch, R. et al., Review: Insulin-dependent DiabetesMellitus, Cell 85:291-297 (1996)). Administration of IL-4 to NOD mice,which serves as an animal model of IDDM, down-regulates the Th1 cellpopulation and significantly delays the onset of diabetes (Rapoport, etal., IL-4 Reverses T cell Proliferation Unresponsiveness and Preventsthe Onset of Diabetes in NOD Mice, J. Exp. Med., 178:87-99 (1993)).Another such autoimmune disease is multiple sclerosis (MS), a diseasewhich is characterized by an autoimmune attack upon the myelin sheathsurrounding nerve cells. Studies in humans with MS have demonstratedthat exacerbation of MS is associated with the presence ofautoantigen-specific Th1 and Th0 cells and that remission is associatedwith the presence of autoantigen-specific Th2 and Th0 cells (Correale, Jet al., Patterns of cytokine secretion by autoreactive proteolipidprotein-specific T cell clones during the course of multiple sclerosis,J. Immunol., 154:2959-2968 (1995)). Mice with experimental autoimmuneencephalomyelitis (EAE), an animal model for MS, also exhibit the Th1cell polarization (Cua, D J, Hinton, D R, and Stohlman, S A, J.Immunol., 155:4052-4059 (1995)). Indirect evidence from a study in theEAE model suggests that IL-4 plays a critical role in diseaseattenuation resulting from treatment with a tolerogenic peptide (Brocke,S. et al. Treatment of experimental encephalomyelitis with a peptideanalogue of myelin basic protein, Nature, 379:343-346 (1996)).

Other autoimmune diseases such as Rheumatoid Arthritis (RA) are alsotargets for IL-4 based therapies. Animal models of RA have shown adisequilibrium of cell profiles tilting towards Th1 cells, and in micethat overexpress TNF-A, anti-TNF-α antibodies have demonstrated diseaseattenuation, suggesting that IL-4 therapies that result indown-regulation of Th1 cell populations may have an anti-TNF-α effectalso. (See Feldmann, M., et al., Review: Rheumatoid Arthritis, Cell,85:307-310 (1996)).

Psoriasis vulgaris is a chronic dermatologic disorder characterized byinfiltration of affected skin with monocytes and T cells. Severalreports indicate that psoriatic skin lesional T cells and PBL arepredominantly of the Th1 phenotype (Uyemura K; Yamamura M, Fivenson D F;Modlin R L; Nickoloff B J, The cytokine network in lesional andlesion-free psoriatic skin is characterized by a T-helper type 1cell-mediated response, J Invest Dermatol., 101:701-705 (1993); SchlaakJ F; Buslau M; Jochum W, Hermann E; Girndt M, Gallati H; Meyer zumBuschenfelde K H; Fleischer B, T cells involved in psoriasis vulgarisbelong to the Th1 subset, J Invest Dermatol, 102:145-149 (1994)).Furthermore, monomethylfumarate, a drug which has been reported to be ofclinical benefit to patients with psoriasis, has been shown toselectively stimulate Th2 cytokine secretion from PBMC (de Jong R;Bezemer A C; Zomerdyk T P; van de Pouw-Kraan T; Ottenhoff T H, NibberingP H, Selective stimulation of T helper 2 cytokine responses by theanti-psoriasis agent monomethylfumarate, Eur J Immunol, 26:2067-2074(1996)). Therefore, IL-4 would be expected to reverse the Thpolarization and be of clinical benefit in psoriasis.

Certain infectious diseases are associated with polarized Th cellresponses to the infectious agent. Th2 responses have in some cases beenassociated with resistance to the infectious agent. An example isBorrelia burgdorfei, the infectious agent for Lyme disease. Humansinfected with B. burgdorferi exhibit a predominantly Th-like cytokineprofile (Oksi J, Savolainen J; Pene J; Bousquet J; Laippala P; ViljanenM K, Decreased interleukin-4 and increased gamma interferon productionby peripheral blood mononuclear cells of patients with Lyme borreliosis,Infect. Immun., 64:3620-3623 (1996)). In a mouse model of B.burgdoreri-induced arthritis, resistance to disease is associated withIL-4 production while susceptibility is associated with INF-γ production(Matyniak J E; Reiner S L, T helper phenotype and genetic susceptibilityin experimental Lyme disease, J Exp Med, 181(3):1251-1254 (1995);Keane-MyersA; Nickell S P, Role of IL-4 and IFN-gamma in modulation ofimmunity to Borrelia burgdorferi in mice, J Immunol 155:2020-2028(1995)). Treatment of B. burgdorferi-infected mice with IL-4 augmentsresistance to the infection (Keane-Myers A; Maliszewski C R; Finkelman FD; Nickell S P, Recombinant IL-4 treatment augments resistance toBorrelia burgdorferi infections in both normal susceptible andantibody-deficient susceptible mice, J Immunol., 156:2488-2494(1996)).

IL-4 has been reported to have a direct effect on inhibiting the growthof lymphomas and leukemias (Akashi, K, The role of interleukin-4 in thenegative regulation of leukemia cell growth, Leuk Lymphoma, 9:205-9(1993)). For example, IL-4 has been reported to induce apoptosis incells from patients with acute lymphoblastic leukemia (Manabe, A, etal., Interleukin-4 induces programmed cell death (apoptosis) in cases ofhigh-risk acute lymphoblastic leukemia, Blood 83:1731-7 (1994)), andinhibits the growth of cells from patients with non-Hodgkin's B celllymphoma (Defrance, T. et al., Antiproliferative effects ofinterleukin-4 on freshly isolated non-Hodgkin malignant B-lymphomacells, Blood, 79:990-6 (1992)).

IL-4 has also been reported to exhibit activities which suggests that itwould be of clinical benefit in osteoarthritis. Osteoarthritis is adisease in which the degradation of cartilage is the primary pathology(Sack, K E, Osteoarthritis, A continuing challenge, West J Med.163:579-86 (1995); Oddis, C V, New perspectives on osteoarthritis, Am JMed. 100:1OS-15S (1996)). IL-4 inhibits TNF-α and IL-1 beta productionby monocytes and synoviocytes from osteoarthritic patients (Bendrups, A,Hilton, A, Meager, A and Hamilton, J A, Reduction of tumor necrosisfactor alpha and interleukin-1 beta levels in human synovial tissue byinterleukin-4 and glucocorticoid, Rheumatol Int, 12:217-20 (1993);Seitz, M et al., Production of interleukin-1 receptor antagonist,inflammatory chemotactic proteins, and prostaglandin E by rheumatoid andosteoarthritic synoviocytes--regulation by IFN-gamma and IL-4, JImmunol, 152:2060-5 (1994)). Additionally, IL-4 has been reported todirectly block the degradation of cartilage in ex vivo cartilageexplants (Yeh, L A, Augustine, A J, Lee, P, Riviere, L R and Sheldon, A,Interleukin-4, an inhibitor of cartilage breakdown in bovine articularcartilage explants, J Rheumatol, 22:1740-6 (1995)). These activitiessuggest that IL-4 would be of clinical benefit in osteoarthritis.

However, the clinical use of IL-4 has been limited due to its acutetoxicity, which is manifested as a vascular leak syndrome (Margolin, K,et al., Phase II Studies of Human Recombinant Interleukin-4 in AdvancedRenal Cancer and Malignant Melanoma, J. Immunotherapy, 15:147-153(1994)). There is no art in the literature which describes the mechanismof the acute toxic effect of IL-4, nor that describes analogs or mutantsof IL-4 that retain immunoregulatory activities but have reduced acutetoxicity.

IL-4 mutant proteins ("muteins") are known. The IL-4 mutein IL-41Y124Dis a T cell antagonist (Kruse N, Tony H P, Sebald W, Conversion of humaninterleukin-4 into a high affinity antagonist by a single amino acidreplacement, Embo J, 11:3237-44 (1992)).

Therapeutic uses of IL-4 found in patents or patent applications includethe following: the use of IL-4 for potentiation of anticancer effects ofchemotherapeutic agents, particularly Hodgkin's Disease and non-HodgkinsLymphoma (see WO 9607422); the use of antigenic fragments of IL-4 togenerate antibodies to treat IL-4 related diseases by suppressing orimitating the binding activity of IL-4 (see WO 9524481), and to detect,measure and immunopurify IL-4 (see WO 9317106); for inducing thedifferentiation of precursor B cells to Immunoglobulin secreting cells,the mature B cells being useful for restoring immune function inimmune-compromised patients (see WO 9404658); when used in combinationwith IL-10, as a therapy for treatment of leukemia, lymphoma,inflammatory bowel disease and delayed type hypersensitivity (e.g.ulcerative colitis and Crohn's Disease)(see WO 9404180); treatment ofHIV infection by administering IL-4 to inhibit viral replication inmonocytes and macrophages, and to increase their cytotoxicity towardssome tumor cells (see WO 9404179); for stimulation of skin fibroblastproliferation for treating wounds in diabetic and immuno-compromisedpatients (see WO 9211861); for enhancing the primary immune responsewhen administering bacterial, toxoid, and viral vaccines, especiallytetanus toxoid vaccine (see WO 9211030); for inhibition of IL-2 inducedproliferation of B cell malignancies, especially chronic lymphocyticleukemia, non-Hodgkin's malignant lymphoma (see WO 9210201); use of IL-4to treat melanomas, renal and basal cell carcinomas (see WO 9204044).

The patent literature discloses IL-4 proteins and some muteins, but nonedirected to an IL-4 therapy with reduced side effects. Lee et al. U.S.Pat. No. 5,017,691 ("the '691 patent") is directed to mammalian proteinsand muteins of human IL-4 which disclose both B-cell growth factoractivity and T cell growth factor activity. It discloses nucleic acidscoding for polypeptides exhibiting IL-4 activity, as well as thepolypeptides themselves and methods for their production. Muteins to thewild-type IL-4 at amino acid positions are disclosed that retain theirability to stimulate both B- and T cell proliferation in vitro. However,nothing in Lee suggests any T cell selective IL-4 muteins, anticipatedactivation of EC's or the endothelial cell leakiness which accompaniesadministration of IL-4. Thus, IL-4 itself is not enabling as atherapeutic modality because of the dose-limiting toxicity.

U.S. Pat. No. 5,013,824 describes hIL-4 peptide derivatives comprisingfrom 6 to 40 amino acids of the native hIL-4. Also disclosed areimmunogens comprising conjugates of the peptides and carriers. Carriersinclude erythrocytes, bacteriophages, proteins, synthetic particles orany substance capable of eliciting antibody production against theconjugated peptide. No muteins of IL-4 are disclosed.

WO96/04306-A2 discloses single-muteins that are antagonists and partialagonists of hIL-2 and hIL-13. No data regarding IL-4 is disclosed.W095/27052 discloses splice mutants of IL-2 and IL-4 containing exons 1,2 and 4.

There exists a need for an improved IL-4 molecule which has reducedtoxicity and is more generally tolerated.

SUMMARY OF THE INVENTION

The invention is directed to human IL-4 muteins numbered in accordancewith wild-type IL-4 having T cell activating activity, but havingreduced endothelial cell activating activity. In particular, human IL-4muteins wherein the surface-exposed residues of the D helix of thewild-type IL-4 are mutated whereby the resulting mutein causes T cellproliferation, and causes reduced IL-6 secretion from HUVECs, relativeto wild-type IL-4. This invention realizes a less toxic IL-4 mutein thatallows greater therapeutic use of this interleukin.

Further, the invention is directed to IL-4 muteins having single, doubleand triple mutations represented by the designators R121A, R121D, R121E,R121F, R121H, R121I, R121K, R121N, R121P, R121T, Rl21W; Y124A, Y124Q,Y124R, Y124S, Y124T; Y124A/S125A, T13D/R121E; and R121T/E122F/Y124Q,when numbered in accordance with wild type IL-4 (His=1). The inventionalso includes polynucleotides coding for the muteins of the invention,vectors containing the polynucleotides, transformed host cells,pharmaceutical compositions comprising the muteins, and therapeuticmethods of treatment.

The invention is also directed to a vector comprising the polynucleotideencoding a mutein of this invention, the vector directing the expressionof a human IL-4 mutein having T cell activating activity but havingreduced endothelial cell activating activity, the vector being capableof enabling transfection of a target organism and subsequent in vivoexpression of said human IL-4 mutein coded for by said polynucleotide.

The invention is also directed to a method of selecting a human IL-4mutein numbered in accordance with wild-type IL-4 having T cellactivating activity but having reduced endothelial cell activatingactivity, comprising mutating the surface-exposed residues of the Dhelix of the wild-type IL-4 whereby the resulting mutein causes T cellproliferation, and causes reduced IL-6 secretion from HUVECs, relativeto wild-type.

The invention is also directed to a method of treating a patientafflicted with an IL-4 treatable condition by administering atherapeutically effective amount of a human IL-4 mutein numbered inaccordance with wild-type IL-4 having T cell activating activity buthaving reduced endothelial cell activating activity. This method isapplicable wherein the IL-4 treatable condition is an autoimmunedisorder, cancer, infectious disease, cartilage disorder, and psoriaticdisorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an amino acid sequence (SEQ ID NO:1) of mature wild-type humanIL-4 used in this study. Helices are underlined and labeled sequentiallyA, B, C, and D. Positions that, when mutated yielded cell-selective IL-4agonists, are indicated in bold type.

FIG. 2 is a graphical presentation of the T cell selective agonistconcept.

FIG. 3 is a composite dose response curve for selective agonist muteinsin the HUVEC IL-6 secretion assay. Panel A:O, wild-type IL-4; ,R121E;∇, R121P;▾, R121T/E122F/Y124Q. Panel B:O, wild-type IL-4; ,Y124Q; ∇, Y124R; ▾, Y124A/S125A.

FIG. 4 are individual dose response curves of selective agonist muteinsin the HUVEC IL-6 secretion assay. Panel A:O, wild-type IL-4; , R121E.Panel B:O, wild-type IL-4; , R121P. Panel C:O, wild-type IL-4; ,Y124Q. Panel D:O, wild-type IL-4; , Y124R. Panel E:O, wild-type IL-4;, Y124A/S125A. Panel F:O, wild-type IL-4; , R12IT/E122F/Y124Q.

FIG. 5 is a composite dose-response curve for selective agonist muteinsfor the biological response of IL-4 muteins in 1° T cell proliferationassays. Panel A:O, wild-type IL-4; , R121E; ∇, R121P; ▾,R121T/E122F/Y124Q. Panel B:O, wild-type IL-4; , Y124Q; ∇, Y124R; ▾,Y124A/S125A.

FIG. 6 are individual dose response curves of the 1° T cellproliferation assay. Panel A:O, wild-type IL-4; , R121E. Panel B:O,wild-type IL-4; , R121P. Panel C:O, wild-type IL-4; , Y124Q. PanelD:O, wild-type IL-4; , Y124R. Panel E:O, wild-type IL-4; ,Y124A/S125A. Panel F:O, wild-type IL-4; , R121T/El22F/Y124Q.

FIG. 7 are individual dose response curves showing the antagonism ofIL-4-induced IL-6 secretion on HUVEC by the T cell-selective agonistIL-4 muteins R121E () and Y124Q (∇). The dose response of the IL-4antagonist R121D/Y124D (0) is included as a control.

FIGS. 8A, 8B are individual dose response curves: Panel A shows thebiological response of the R121D mutein in a 1° T cell proliferationassay (O=IL-4,=R121D); Panel B shows the inability of R121D to induceIL-6 secretion on HUVEC (O=IL-4, =R121D).

FIG. 9A are the individual dose response curves for IL-4 (O) and the Tcell-selective agonist muteins R121E (∇) and T13D/R121E (▴) in the 1° Tcell proliferation assay.

FIG. 9B are individual dose response curves showing the antagonism ofIL-4-induced IL-6 secretion on HUVEC by the T cell-selective IL-4agonist muteins R121E (Δ) and T13D/R121E (▴).

DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Background

IL-4 has been shown to mediate a variety of cellular responses in vitro,including various effects on B cells, T cells, and monocytes, as well asendothelial cells (Maher D W, Davis I, Boyd A W, Morstyn G: Humaninterleukin-4: an immunomodulator with potential therapeuticapplications. Prog Growth Factor Res 3:43-56, 1991; Powrie F, Coffman RL: Cytokine regulation of T cell function: potential for therapeuticintervention. Immunol Today 14:270-4, 1993). In particular, upregulationof vascular cell adhesion molecule-1 (VCAM-1; (Swerlick R A, Lee K H, LiL J, Sepp N T, Caughmnan S W, Lawley T J: Regulation of vascular celladhesion molecule I on human dermal microvascular endothelial cells. JImmunol 149:698-705, 1992)) and induction of IL-6 (Colotta F, Sironi M,Borre A, Luini W, Maddalena F, Mantovani A: Interleukin 4 amplifiesmonocyte chemotactic protein and interleukin 6 production by endothelialcells. Cytokine 4:24-8, 1992) and monocyte chemoattractant protein-1(MCP-1; Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A:Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6production by endothelial cells. Cytokine 4:24-8, 1992; Rollins B J,Pober J S: Interleukin-4 induces the synthesis and secretion of MCP-1/JEby human endothelial cells. Am J Pathol 138:1315-9, 1991)) are directeffects of IL-4 on cultured endothelial cells; the upregulation ofVCAM-1 is correlated with the increased adhesion of lymphocytes both invitro (Carlos T M, Schwartz B R, Kovach N L, Yee E, Rosa M, Osborn L,Chi-Rosso G, Newman B, Lobb R, Rosso M, et al.: Vascular cell adhesionmolecule-1 mediates lymphocyte adherence to cytokine-activated culturedhuman endothelial cells. Blood 76:965-70, 1990; Thornhill M H, WellicomeS M, Mahiouz D L, Lanchbury J S, Kyan-Aung U, Haskard D O: Tumornecrosis factor combines with IL-4 or IFN-gamma to selectively enhanceendothelial cell adhesiveness for T cells. The contribution of vascularcell adhesion molecule-1-dependent and -independent binding mechanisms.J Immunol 146:592-8, 1991) and in vivo (Briscoe D M, Cotran R S, Pober JS: Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on theexpression of vascular cell adhesion molecule-1 in vivo. Correlationwith CD3+T cell infiltration. J Immunol 149:2954-60, 1992).

The IL-4 mutein IL-4/Y124D (substitution of Aspartic acid for Tyrosineat position 124) is a T cell antagonist (Kruse N, Tony H P, Sebald W:Conversion of human interleukin-4 into a high affinity antagonist by asingle amino acid replacement. Embo J 11:3237-44, 1992). In vivoexperiments performed by the inventors have demonstrated that IL-4/Y124Dexhibits acute toxicity similar to that of wild-type IL-4 in monkeys, apreviously undescribed observation. The cellular events associated withboth wild-type IL-4- and IL-4/Y124D-mediated toxicity includeupregulation of VCAM-1, upregulation of MCP-1 in serum, increases incirculating monocytes together with a concomitant decrease incirculating lymphocytes, and an increase in hematocrit. Similar cellulartrafficking has been observed in clinical trials using IL-4 in humans(Wong H L, Lotze M T, Wahl L M, Wahl S M: Administration of recombinantIL-4 to humans regulates gene expression, phenotype, and function incirculating monocytes. J Immunol 148:2118-25, 1992). Due to itsproperties as an antagonist of T cells, these results suggest that thetoxicities demonstrated by IL-4/Y124D are due to agonist activities onand are mediated through cells other than T cells. The observed in vivotoxicities using IL-4/Y124D and the known effects of IL-4 on endothelialcells are consistent with the mechanism that in vivo IL-4 toxicity ismediated through direct effects of IL-4 on the vascular endothelium.

Through these (and related) investigations, the inventors havediscovered that a new IL-4 receptor may exist on endothelial cells("EC"). This possibility led to efforts to synthesize IL-4 muteins thatwould selectively activate T cells, but not EC. While T cells express anIL-4 receptor composed of IL-4Rα and IL-2Rγ subunits, the inventors havediscovered that human umbilical vein endothelial cells (HUVEC), expressIL-4Rα but not IL-2Rγ. Crosslinking studies have shown that two receptorchains are expressed at the cell surface of HUVEC: the molecular weightof one is consistent with IL-4Rα, and a second, lower molecular weightchain. These results suggest that a novel IL-4 receptor componentsimilar in function to IL-2Rγ, but differing in sequence, is expressedon HUVECs. The differences in the specific molecular structures betweenthese two receptors were thus exploited to generate an IL-4 variant thatis selective for one receptor over the other (e.g., a T cell selectiveagonist).

FIG. 2 demonstrates graphically the selective agonist concept. It showsthe T cell IL-4 receptor comprising the IL-4Rα/IL-2Rγ subunit, and anEndothelial cell IL-4 receptor comprising the IL-4Rαb/γ-like receptorsubunit. Although depicted here together for purposes of illustrationonly, the two receptors for IL-4 are expressed on different cell types.The T cell receptor is composed of IL-4Rα and IL-2Rγ; IL-4 bindinginduces receptor heterodimer formation that results in cellularsignalling. IL-4 induced receptor heterodimer formation occurs in asimilar manner on EC's, except that the receptor for IL-4 is composed ofIL-4Rα and a yHike receptor component. The tγ-like receptor component isdifferent from IL-2Rγ. T cell-selective IL-4 agonists are those variantsof IL-4 that retain their ability to interact with the T cell receptorIL-4Rox/IL-2Rγ, but are unable to induce heterodimerization, and thussignalling, of the non-T cell receptor IL-4Rα/γ-like subunit. Such Tcell-selective IL-4 agonists retain their ability to interact withIL-4Rα; it is their ability to discriminate between IL-2Rγ and theγ-like subunit that gives them their cell-selective activationproperties.

The two components of the T cell receptor, IL4-Rα and IL-2Rγ, contactdifferent regions of the IL-4 molecule, and therefor the inventors havefocussed on a small region of IL-4 to modify. Hypothesizing that thenovel receptor subunit would contact the same region of IL4 as doesIL-2Rγ, the inventors made a number of substitutions in the D-Helix,particularly residues 121, 124 and 125.

The D-helix has been implicated in interactions with both IL-2Rγ andwith the putative novel receptor on HUVEC (specifically, the IL-4 muteinR121D/Y124D is a HUVEC antagonist). Muteins containing modifications tothe D-helix of IL-4 (residues 110 to 126; His=1) were screened for theirability to stimulate either T cell proliferation or human umbilical veinendothelial cell (HUVEC) secretion of IL-6. Muteins that induced adifferential response on T cells relative to HUVEC were furthercharacterized through further mutagenesis.

An initial scan of the D helix was undertaken to determine the potentialareas of interaction. Additionally, alanine scanning substitutions ofthe AB loop were also generated, as this region is suggested to beinvolved in the interaction of the cytokine ligand and the D-helixinteracting receptor subunit. In particular, surface-exposed residuesGlu- 110, Asn-111, Glu-1 14, Arg-l 15, Lys-117, Thr-1 18, Arg-121,Glu-122, Tyr-124, Ser-125, and Lys-126 were targeted for investigationand are preferred targets for mutation analysis. Sites 118-126 are morepreferred, and sites 121-125 are most preferred. Comparisons betweenIL-2, IL-4, IL-7 and IL-15 in this region also identify differencesbetween IL-4 and IL-2, IL-7 and IL-15, possibly suggesting specificresidues responsible for the HUVEC receptor interaction. Specificsubstitutions derived from an alignment between IL-2 and IL-4 wereintroduced into IL-4. These included: Arg-1 15 to Phe; Lys-1 17 to Asn;Glu-122 to Phe; Lys-126 to Ile; and three simultaneous changes Arg-121to Thr, Glu-122 to Phe, and Tyr-124 to Gln.

Mutations were introduced using site-directed mutagenesis on wild-typehuman IL-4 cDNA. Correct clones were subdloned to an expression vectorsuitable for expression in a heterologous system (e.g., E. coli,baculovirus, or CHO cells). Purified proteins were tested in T cellproliferation and HUVEC cytokine secretion assays (IL-6). Differentresponses generated by individual muteins between these assays, eitherin EC₅₀ or maximal response (plateau) indicate mutations that effectthese activities. Specifically, muteins that stimulate a relativelystronger response in the T cell assay (vs. wild-type IL-4) as comparedto the response on HUVEC (vs. wild-type IL-4) will suggest positionsthat are more important to the interaction of IL-4 with IL-2Rγ than theinteraction of IL-4 with the novel HUVEC IL-4 receptor. Further analysisand mutagenesis (e.g. combinatorial changes, substitution with all aminoacids) of the identified positions will produce an IL-4 mutein withselective agonist properties for the T cell IL-4 receptor. This proteinwill also be a selective antagonist for IL-4-induced HUVEC responses.

B. Definitions

Described herein are novel muteins and a mechanism for deriving novelIL-4 muteins with selective agonist properties on T cells and reducedtoxicity. A similar strategy may be used to identify a T cell-selectiveantagonist.

As used herein, "wild type IL-4" means IL-4, whether native orrecombinant, having the 129 normally occurring amino acid sequence ofnative human IL-4, as shown, e.g., in FIG. 1.

As used herein, "IL-4 mutein" means a polypeptide wherein specificsubstitutions to the human mature interleukin-4 protein have been made.Specifically disclosed herein, the arginine residue (R) at position 121("Arg-121"), when numbered in accordance with wild type IL-4, issubstituted with alanine (A), aspartate (D), glutamate (E),phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine(N) proline (P), threonine (T) or tryptophan (W); or the glutamate (E)residue at position 122 is substituted with phenylalanine (F); or thetyrosine residue at position 124 is substituted with alanine (A),glutamine (Q), arginine (R) serine (S) or threonine (T); or the serine(S) residue at position 125 is substituted with alanine (A). Our mostpreferred IL-4 muteins have an amino acid sequence identical to wildtype IL-4 at the other, non-substituted residues. However, the IL-4muteins of this invention may also be characterized by amino acidinsertions, deletions, substitutions and modifications at one or moresites in or at the other residues of the native IL-4 polypeptide chain.In accordance with this invention any such insertions, deletions,substitutions and modifications result in an IL-4 mutein that retains aT cell-selective activity while having reduced ability to activateendothelial cells.

We prefer conservative modifications and substitutions at otherpositions of IL-4 (i.e., those that have a minimal effect on thesecondary or tertiary structure of the mutein). Such conservativesubstitutions include those described by Dayhoff in The Atlas of ProteinSequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785(1989). For example, amino acids belonging to one of the followinggroups represent conservative changes:

ala, pro, gly, gln, asn, ser, thr;

cys, ser, tyr, thr;

val, ile, leu, met, ala, phe;

lys, arg, his;

phe, tyr, trp, his; and

asp, glu.

We also prefer modifications or substitutions that do not introducesites for additional intermolecular crosslinking or incorrect disulfidebond formation. For example, IL-4 is known to have six cys residues, atwild-type positions 3, 24, 46, 65, 99 and 127.

By "numbered in accordance with wild type IL-4" we mean identifying achosen amino acid with reference to the position at which that aminoacid normally occurs in wild type IL-4. Where insertions or deletionsare made to the IL-4 mutein, one of skill in the art will appreciatethat the ser (S) normally occurring at position 125, when numbered inaccordance with wild type IL-4, may be shifted in position in themutein. However, the location of the shifted ser (S) can be readilydetermined by inspection and correlation of the flanking amino acidswith those flanking ser in wild type IL-4.

The IL-4 muteins of the present invention can be produced by anysuitable method known in the art. Such methods include constructing aDNA sequence encoding the IL-4 muteins of this invention and expressingthose sequences in a suitably transformed host. This method will producerecombinant muteins of this invention. However, the muteins of thisinvention may also be produced, albeit less preferably, by chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

In one embodiment of a recombinant method for producing a mutein of thisinvention, a DNA sequence is constructed by isolating or synthesizing aDNA sequence encoding the wild type IL-4 and then changing the codon forarg121 to a codon for alanine (A), aspartate (D), glutamate (E),phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine(N) proline (P), threonine (T) or tryptophan (W) by site-specificmutagenesis. This technique is well known. See, e.g., Mark et al.,"Site-specific Mutagenesis Of The Human Fibroblast Interferon Gene",Proc. Natl. Acad. Sci. USA 81, pp. 5662-66 (1984); U.S. Pat. No.4,588,585, incorporated herein by reference.

Another method of constructing a DNA sequence encoding the IL-4 muteinsof this invention would be chemical synthesis. For example, a gene whichencodes the desired IL-4 mutein may be synthesized by chemical meansusing an oligonucleotide synthesizer. Such oligonucleotides are designedbased on the amino acid sequence of the desired IL-4 mutein, andpreferably selecting those codons that are favored in the host cell inwhich the recombinant mutein will be produced. In this regard, it iswell recognized that the genetic code is degenerate--that an amino acidmay be coded for by more than one codon. For example, phe (F) is codedfor by two codons, TTC or TTT, tyr (Y) is coded for by TAC or TAT andhis (H) is coded for by CAC or CAT. Trp (W) is coded for by a singlecodon, TGG. Accordingly, it will be appreciated that for a given DNAsequence encoding a particular IL-4 mutein, there will be many DNAdegenerate sequences that will code for that IL-4 mutein. For example,it will be appreciated that in addition to the preferred DNA sequencefor mutein R121E shown in SEQ ID NO:3, there will be many degenerate DNAsequences that code for the IL-4 mutein shown. These degenerate DNAsequences are considered within the scope of this invention. Therefore,"degenerate variants thereof" in the context of this invention means allDNA sequences that code for a particular mutein.

The DNA sequence encoding the IL-4 mutein of this invention, whetherprepared by site directed mutagenesis, synthesis or other methods, mayor may not also include DNA sequences that encode a signal sequence.Such signal sequence, if present, should be one recognized by the cellchosen for expression of the IL-4 mutein. It may be prokaryotic,eukaryotic or a combination of the two. It may also be the signalsequence of native IL-4. The inclusion of a signal sequence depends onwhether it is desired to secrete the IL-4 mutein from the recombinantcells in which it is made. If the chosen cells are prokaryotic, itgenerally is preferred that the DNA sequence not encode a signalsequence. If the chosen cells are eukaryotic, it generally is preferredthat a signal sequence be encoded and most preferably that the wild-typeIL-4 signal sequence be used.

Standard methods may be applied to synthesize a gene encoding an IL-4mutein according to this invention. For example, the complete amino acidsequence may be used to construct a back-translated gene. A DNA oligomercontaining a nucleotide sequence coding for IL-4 mutein may besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and then ligated.The individual oligonucleotides typically contain 5' or 3' overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the DNA sequences encoding an IL-4 mutein of this inventionwill be inserted into an expression vector and operatively linked to anexpression control sequence appropriate for expression of the IL-4mutein in the desired transformed host. Proper assembly may be confirmedby nucleotide sequencing, restriction mapping, and expression of abiologically active polypeptide in a suitable host. As is well known inthe art, in order to obtain high expression levels of a transfected genein a host, the gene must be operatively linked to transcriptional andtranslational expression control sequences that are finctional in thechosen expression host.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations may be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including colEl, pCRl, pER32z, pM9 and their derivatives, wider host range plasmids,such as RP4, phage DNAs, e.g., the nurnerous derivatives of phagelambda, e.g., NM989, and other DNA phages, such as M13 and filamentoussingle stranded DNA phages. Useful expression vectors for yeast cellsinclude the 2 μ plasmid and derivatives thereof. Useful vectors forinsect cells include pVL 941. We prefer pFastBac™ 1 (GibcoBRL,Gaithersburg, Md.). Cate et al., "Isolation Of The Bovine And HumanGenes For Mullerian Inhibiting Substance And Expression Of The HumanGene In Animal Cells", Cell, 45, pp. 685-98 (1986).

In addition, any of a wide variety of expression control sequences maybe used in these vectors. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol seguences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the trp system, the TAC or TRCsystem, the major operator and promoter regions of phage lambda, forexample PL, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., PhoA, the promoters of the yeast x-matingsystem, the polyhedron promotor of Baculovirus, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells or their viruses, and various combinations thereof.

Any suitable host may be used to produce the IL-4 muteins of thisinvention, including bacteria, fingi (including yeasts), plant, insect,mammal, or other appropriate animal cells or cell lines, as well astransgenic animals or plants. More particularly, these hosts may includewell known eukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such asSpodoptera frugiperda (SJ9), animal cells such as Chinese hamster ovary(CHO) and mouse cells such as NS/O, African green monkey cells such asCOS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well asplant cells in tissue culture. For animal cell expression, we prefer CHOcells and COS 7 cells in cultures and particularly the CHO cell line CHO(D HFR-).

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences described herein. Neither will all hosts fimction equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, preferredvectors for use in this invention include those that allow the DNAencoding the IL-4 muteins to be amplified in copy number. Suchamplifiable vectors are well known in the art. They include, forexample, vectors able to be amplified by DHFR amplification (see, e.g.,Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of AModular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals UtilizedFor Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) orglutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No.5,122,464 and European published application 338,841).

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the actualDNA sequence encoding the IL-4 mutein of this invention, particularly asregards potential secondary structures. Hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the DNA sequences of thisinvention, their secretion characteristics, their ability to fold thepolypeptides correctly, their fermentation or culture requirements, andthe ease of purification of the products coded for by the DNA sequences.

Within these parameters, one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe desired DNA seguences on fermentation or in large scale animalculture, for example, using CHO cells or COS 7 cells.

The IL-4 muteins obtained according to the present invention may beglycosylated or unglycosylated depending on the host organism used toproduce the mutein. If bacteria are chosen as the host then the IL-4mutein produced will be unglycosylated. Eukaryotic cells, on the otherhand, will glycosylate the IL-4 muteins, although perhaps not in thesame way as native IL-4 is glycosylated.

The IL-4 mutein produced by the transformed host can be purifiedaccording to any suitable method. Various methods are known forpurifying IL-4. See, e.g., U.S. Pat. Nos. 5,013,824; 5,017,691; andWO9604306-A2. We prefer immunoaffinity purification. See, e.g., Okamuraet al., "Human Fibroblastoid Interferon: Immunosorbent ColumnChromatography And N-Terminal Amino Acid Sequence", Biochem., 19, pp.3831-35 (1980).

The biological activity of the IL-4 muteins of this invention can beassayed by any suitable method known in the art. Such assays includeantibody neutralization of antiviral activity, induction of proteinkinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities,as described in EP-B1-41313. Such assays also include immunomodulatoryassays (see, e.g., U.S. Pat. No. 4,753,795), growth inhibition assays, Tcell proliferation, induction of IL-6 (MCP-l or VCAM-1) on EC andmeasurement of binding to cells that express interleukin-4 receptors.See also Spits H, Yssel H, Takebe Y, et al., Recombinant Interleukin-4Promotes the Growth of Human T Cells, J. IMMUNOL 139:1142-47 (1987).

The IL-4 mutein of this invention will be administered at a doseapproximately paralleling that or greater than employed in therapy withwild type native or recombinant IL-4. An effective amount of the IL-4mutein is preferably administered. An "effective amount" means an amountcapable of preventing or lessening the severity or spread of thecondition or indication being treated. It will be apparent to those ofskill in the art that the effective amount of IL-4 mutein will depend,inter alia, upon the disease, the dose, the administration schedule ofthe IL-4 mutein, whether the IL-4 mutein is administered alone or inconjunction with other therapeutic agents, the serum half-life of thecomposition, and the general health of the patient.

The IL-4 mutein is preferably administered in a composition including apharmaceutically acceptable carrier. "Pharmaceutically acceptablecarrier"means a carrier that does not cause any untoward effect inpatients to whom it is administered. Such pharmaceutically acceptablecarriers are well known in the art. We prefer 2% HSA/PBS at pH 7.0.

The IL-4 muteins of the present invention can be formulated intopharmaceutical compositions by well known methods. See, e.g.,Remington's Pharmacautical Science by E. W. Martin, hereby incorporatedby reference, describes suitable formulations. The pharmaceuticalcomposition of the IL-4 mutein may be formulated in a variety of forms,including liquid, gel, lyophilized, or any other suitable form. Thepreferred form will depend upon the particular indication being treatedand will be apparent to one of skill in the art.

The IL-4 mutein pharmaceutical composition may be administered orally,by aerosol, intravenously, intramuscularly, intraperitoneally,intradermally or subcutaneously or in any other acceptable manner. Thepreferred mode of administration will depend upon the particularindication being treated and will be apparent to one of skill in theart. The pharmaceutical composition of the IL-4 mutein may beadministered in conjunction with other therapeutic agents. These agentsmay be incorporated as part of the same pharmaceutical composition ormay be administered separately from the IL-4 mutein, either concurrentlyor in accordance with any other acceptable treatment schedule. Inaddition, the IL-4 mutein pharmaceutical composition may be used as anadjunct to other therapies.

Accordingly, this invention provides compositions and methods fortreating immune disorders, cancers or tumors, abnormal cell growth, orfor immunomodulation in any suitable animal, preferably a mammal, mostpreferably human. As previously noted in the Background section, IL-4has many effects. Some of these are stimulation of T cell proliferation,T-helper cell differentiation, induction of human B-cell activation andproliferation, and lymphokine-directed immunoglobulin class switching.Effects on the lymphoid system include increasing the expression of MHCclass II antigen (Noelle, R., et al., Increased Expression of IaAntigens on resting B cells: a New Role for B Cell Growth Factor, PNASUSA, 81:6149-53 (1984)), and CD 23 on B cells (Kikutani, H., et al.,Molecular Structure of Human Lymphocyte Receptor for Immunoglobulin,Cell 47:657-61 (1986)). T-helper cell type 1 (Th1 ) and type 2 (Th2) areinvolved in the immune response. Stimulated Th2 cells secrete IL-4 andblock Th1 progression. Thus, any Th1-implicated disease is amenable totreatment by IL-4 or analogs thereof.

Also contemplated is use of the DNA sequences encoding the IL-4 muteinsof this invention in gene therapy applications. Gene therapyapplications contemplated include treatment of those diseases in whichIL-4 is expected to provide an effective therapy due to itsimmunomodulatory activity, e.g., Multiple Sclerosis (MS),Insulin-dependent Diabetes Mellitus (IDDM), Rheumatoid Arthritis (RA),Systemic Lupus Erythematosus (SLE), uveitis, orchitis, primary biliarycirrhosis, malaria, leprosy, Lyme Disease, contact dermatitis,psoriasis, B cell lymphoma, acute lymphoblastic leukemia, non-Hodgkinslymphoma, cancer, osteoarthritis and diseases that are otherwiseresponsive to IL-4 or infectious agents sensitive to IL-4-mediatedimmune response.

Local delivery of IL-4 muteins using gene therapy may provide thetherapeutic agent to the target area. Both in vitro and in vivo genetherapy methodologies are contemplated. Several methods for transferringpotentially therapeutic genes to defined cell populations are known.See, e.g., Mulligan, "The Basic Science Of Gene Therapy", Science, 260:926-31 (1993). These methods include:

1) Direct gene transfer. See, e.g., Wolff et al., "Direct Gene transferInto Mouse Muscle In Vivo", Science, 247:1465-68 (1990);

2) Liposome-mediated DNA transfer. See, e.g., Caplen at al.,"Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium OfPatients With Cystic Fibrosis", Nature Med. 3: 39-46 (1995); Crystal,"The Gene As A Drug", Nature Med. 1:15-17 (1995); Gao and Huang, "ANovel Cationic Liposome Reagent For Efficient Transfection Of MammalianCells", Biochem. Biophys. Res. Comm., 179:280-85 (1991);

3) Retrovirus-mediated DNA transfer. See, e.g., Kay et al., "In VivoGene Therapy Of Hemophilia B: Sustained Partial Correction In FactorIX-Deficient Dogs", Science 262:117-19 (1993); Anderson, "Human GeneTherapy", Science, 256:808-13 (1992).

4) DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably "defective" or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). See, e.g., Ali et al., "The Use Of DNA Viruses AsVectors For Gene Therapy", Gene Therapy, 1:367-84 (1994); U.S. Pat. No.4,797,368, incorporated herein by reference, and U.S. Pat. No.5,139,941, incorporated herein by reference.

The choice of a particular vector system for transferring the gene ofinterest will depend on a variety of factors. One important factor isthe nature of the target cell population. Although retroviral vectorshave been extensively studied and used in a number of gene therapyapplications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity.

Adenoviruses have the advantage that they have a broad host range, caninfect quiescent or terminally differentiated cells, such as neurons orhepatocytes, and appear essentially non-oncogenic. See, e.g., Ali etal., supra, p. 367. Adenoviruses do not appear to integrate into thehost genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Ali et al., supra, p. 373.

Adeno-associated viruses exhibit similar advantages as adenoviral-basedvectors. However, AAVs exhibit site-specific integration on humanchromosome 19. Ali et al., supra, p. 377.

In a preferred embodiment, the IL-4 mutein-encoding DNA of thisinvention is used in gene therapy for autoimmune diseases such as MS,IDDM, and RA, infectious diseases such as Lyme Disease and Leprosy,cancers, such as non-Hodgkins lymphoma and ALL, cartiledgenous disorderssuch as osteoarthritis, and psoriatic conditions, such as psoriasis.

According to this embodiment, gene therapy with DNA encoding the IL-4muteins of this invention is provided to a patient in need thereof,concurrent with, or immediately after diagnosis.

This approach takes advantage of the selective activity of the IL-4muteins of this invention to prevent undesired autoimmune stimulation.The skilled artisan will appreciate that any suitable gene therapyvector containing IL-4 mutein DNA may be used in accordance with thisembodiment. The techniques for constructing such a vector are known.See, e.g., Ohno et al., supra, p. 784; Chang et al., supra, p. 522.Introduction of the IL-4 mutein DNA-containing vector to the target sitemay be accomplished using known techniques, e.g., as described in Ohnoet al., supra, p. 784.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only, and are not to be construed as lirmiting the scope ofthe invention in any manner.

EXAMPLES Generally

The amino acid sequence of mature human IL-4 used in this study is shownbelow. Amino acids at which substitutions yielded T cell selectiveagonists are indicated in bold type:

    __________________________________________________________________________    His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn                   1               5                   10                  15                    Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr                                   20                  25                  30                    Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe                                   35                  40                  45                    Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu                                   50                  55                  60                    Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg                                   65                  70                  75                    His Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu                                   80                  85                  90                    Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala Asn                                   95                  100                 105                   Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met                                   110                 115                 120                   Arg Glu Lys Tyr Ser Lys Cys Ser Ser               (SEQ ID NO:1)                               125                                                           __________________________________________________________________________

Muteins were expressed in a baculovirus system, purified to homogeneity,and evaluated in biological assays that reflected different IL-4receptor usage. Two assays were used to test for selective agonistactivity, IL-4-induced HUVEC IL-6 secretion assay, and 1° T cellproliferation assay for positive IL-4 activity. Compounds that have theability to induce 1° T cell proliferation, yet have a reduced ability toinduce IL-6 secretion, are T cell selective IL-4 agonists and comewithin the scope of this invention. More specifically, human umbilicalvein endothelial cells (HUVEC) were used to assess activity through thealternate IL-4 receptor (IL-4Rα/γ-like receptor component).

Example 1 Production of Muteins

Muteins were generated by site-directed mutagenesis using primerscontaining codons corresponding to the desired mutation essentially asdescribed by Kunkel T A, Roberts J D, and Zakour R A, "Rapid andefficient site-specific mutagenesis without phenotypic selection"(1987),Methods Enzymol 154: 367-382. Briefly, human IL-4 cDNA containing therestricition enzyme sites Bam HI and Xba I was subdloned into the M13phage vector M13 mp19 (New England Biolabs, Beverly, Mass.) using thesame sites. Wild-type IL-4 cDNA was obtained using Polymerase ChainReaction ("PCR") from a cDNA pool generated from mRNA isolated fromhuman peripheral blood lymphocytes induced 24 hours with phorbol12-myristate 13-acetate (10 ng/ml). The PCR primers used were, for the5'end of the IL-4 open reading frame,

    5'-CGC GGA TCC ATG GGT CTC ACC TCC-3'(SEQ ID NO:22);

and for the 3' end of the IL-4 open reading frame,

    5'-CGC TCT AGA CTA GCT CGA ACA CTT TGA AT-3'(SEQ ID NO:23).

Restriction enzyme sites BamHI (5'-end) and XbaI (3'-end) wereincorporated into each oligonucleotide and are indicated by italics. ThePCR conditions used were 1 minute at 94° C., 1 minute at 58.7° C., and 1minute at 72° C. for 25 cycles. The correct IL-4 cDNA sequence soobtained was confirmed by sequencing using the Sequenase® sequencing kit(Amersham Life Sciences, Arlington Heights, Ill.) as described by themanufacturer. Uracil-containing single strand DNA (U-DNA) was obtainedby transforming the E. coli strain CJ236 (Bio-Rad Laboratories,Hercules, Calif.) with IL-4 cDNA-containing M13 mpl9. Site-directedmutagenesis utilized in general primers containing 15 nucleotideshomologous to the template U-DNA 5' to the codon(s) targetted formutagnesis, nucleotides that incorporate the desired change, and anadditional 10 nucleotides homologous to the template U-DNA 3' of thelast altered nucleotide. The specific primers used were:

    __________________________________________________________________________    R121A:     CTAAAGACGA TCATGGCTGA GAAATATT                                                                       (SEQ ID NO:24)                              R121D:          GCTAAAGACG ATCATGGACG AGAAATATTC                                                                        (SEQ ID NO:25)                      R121E:          GCTAAAGACG ATCATGGAAG AGAAATATTC                                                                        (SEQ ID NO:26)                      R121F:          CTAAAGACGA TCATGTTTGA GAAATATT                                                                            (SEQ ID NO:27)                    R121H:          CTAAAGACGA TCATGCACGA GAAATATT                                                                            (SEQ ID NO:28)                    R121I:          CTAAAGACGA TCATGATAGA GAAATATT                                                                            (SEQ ID NO:29)                    R121K:          CTAAAGACGA TCATGAAAGA GAAATATT                                                                            (SEQ ID NO:30)                    R121N:          CTAAAGACGA TCATGAACGA GAAATATT                                                                            (SEQ ID NO:31)                    R121P:          GCTAAAGACG ATCATGCCAG AGAAATATTC                                                                        (SEQ ID NO:32)                      R121T:          CTAAAGACGA TCATGACTGA GAAATATT                                                                            (SEQ ID NO:33)                    R121W:          CTAAAGACGA TCATGTGGGA GAAATATT                                                                            (SEQ ID NO:34)                    Y124A:          ATCATGAGAG AGAAAGCATC AAAGTGTT                                                                            (SEQ ID NO:3S)                    Y124Q:          ATCATGAGAG AGAAACAATC AAAGTGTT                                                                            (SEQ ID NO:36)                    Y124R:          ATCATGAGAG AGAAACGATC AAAGTGTT                                                                            (SEQ ID NO:37)                    Y124S:          ATCATGAGAG AGAAATCATC AAAGTGTT                                                                            (SEQ ID NO:38)                    Y124T:          ATCATGAGAG AGAAAACATC AAAGTGTT                                                                            (SEQ ID NO:39)                    Y124A/S125A:                                                                             CGATCATGAG AGAGAAAGCT GCTAAGTGTT CGA                                                                      (SEQ ID NO:40)                         T13D:            CAGGAGATCA TCAAAGATTT GAACAGCC                                                                           (SEQ ID NO:41)                    R121T/E122F/Y124Q:                                                                       GCTAAAGACG ATCATGACCT TCAAACAGTC AAAG                                                                (SEQ ID NO:42)                              __________________________________________________________________________

Regions of mutated nucleotides are underlined. Primers werephosphorylated using T4 polynucleotide kinase (New England Biolabs,Beverly, MA) using the manufacturer's protocol. After annealing of theprimer to the U-DNA template and extension with T7 DNA polymerase(Bio-Rad Laboratories, Hercules, Calif.), cells of the E. coli strainDH5α™ (GibcoBRL, Gaithersburg, Md.) were transformed with 5 μl ofreaction mixture and plated in LB medium containing 0.7% agar. Afterincubation at 37° C., plaques were expanded by picking a single plaqueand transferring to 2 mls of LB media and grown overnight at 37° C.Single strand DNA was isolated using an M13 purification kit (Qiagen,Inc., Chatsworth, Calif.) per manufacturer's protocol, and clonescontaining the desired mutation were identified by sequencing the singlestranded DNA using the Sequenase® sequencing kit (Amersham LifeSciences, Arlington Heights, Ill.) per manufacturer's protocol. IL-4mutein cDNA from Replicative Form DNA corresponding to plaquescontaining the correct mutated sequence was isolated using Bam HI andXba I, and subcloned to the plasmid vector pFastBac™ 1 (GibcoBRL,Gaithersburg, Md.). After subdloning, recombininant baculovirus DNA(hereafter referred to as Bacmid) was generated by transformingpFastBac™ 1 containing the mutein cDNA to the E. coli strain DHlOBac™(GibcoBRL, Gaithersburg, Md.) as described by the manufacturer. Muteinswere expressed in Spodoptera ftugiperda (SJ) 9 cells using theBac-to-Bac (GibcoBRL, Gaithersburg, Md.) baculovirus expression system.All insect cell incubations occurred at 28° C. Briefly, 2 ml cultures ofSf 9 cells were transfected with 5μl of recombinant Bacmid usingCelIFECTIN (GibcoBRL, Gaithersburg, Md.). The supernatant was harvested60 hours post-transfection, and used to infect a 100-200 ml culture of1×10⁶ Sf 9 cells/ml in Grace's media (GibcoBRL, Gaithersburg, Md.). Permanufacturer's protocol, the supernatants were harvested 48-60 hrspost-infection by centrifugation at 5000 rpm for 10 minutes in aSorvall® RC-5B centrifuge using a GSA rotor (Dupont Instrument Co.,Wilmington, Del.) and assayed for virus titre (typically, >1×10⁸ plaqueforming units/ml was obtained). For protein production, 2-3×10⁶ Sf9cells/ml in 500 mls of SF900 II media (GibcoBRL, Gaithersburg, Md.) wereinfected at a multiplicity of infection between 4-10 and the supernatantwas harvested 60-72 hrs post-infection by centrifugation at 5000 rpm for10 minutes in a Sorvall® RC-5B centrifuge using a GSA rotor (DupontInstrument Co., Willmington, Del.) and filtered through a sterile 0.2 μMfilter unit.

Example 2 Purification of Muteins

Anti-human IL-4 monoclonal antibodies C400.1 and C400.17 were generatedusing standard protocols from mice using recombinant human IL-4 (GenzymeDiagnostics, Cambridge, Mass.) as immunogen, were produced as ascitesfluid, purified, and coupled to CNBr-activated Sepharose (Pharmacia,Uppsala, Sweden) as per manufacturer's protocol. Sf 9 cell supernatantsgenerated from infection of Sf 9 cells by recombinant baculoviruscontaining the respective IL-4 mutein were loaded onto a 1 ml column ofIL-4 affinity matrix, washed with 100 mM NaHCO₃, 500 mM NaCl, pH 8.3,washed with water to remove salt, and eluted with 8 column volumes of100 mM Glycine, pH 3.0. Fractions were collected in siliconized vialscontaining 0.1 volume 1 M Tris, pH 8.0. Mutein protein was furtherpurified by reverse phase chromatography using a Dynarnax®-300 Å C ₁₈column (Rainin Instrument Co., Woburn, Mass.) with a 0.1% gradient ofBuffer A to B (Buffer A, water; Buffer B, acetonitrile, 0.1%trifluoroacetic acid). Fractions were evaluated by SDS-PAGE, and muteincontaining fractions were lyophilized for storage, and resuspended insterile phosphate-buffered saline for assays. Mutein so purified wastypically a single band as observed by SDS-PAGE (silver stain), and wasquantitated by amino acid analysis (accuracy typically >90%).

Example 3 1° T Cell Proliferation Assay

Primary T cells were obtained from fresh blood from normal donors andpurified by centrifugation using Ficoll-Paque® Plus (Pharmacia, Upsalla,Sweden) essentially as described by Kruse, N., Tony, H. P. and Sebald,W. "Conversion of human interleukin-4 into a high affinity antagonist bya single amino acid replacement", Embo J 11: 3237-44 (1992). Thepurified peripheral blood mononuclear cells were incubated for 7 dayswith 10 gg/ml phytohemagglutinin (Sigma Chemical Co., St. Louis, Mo.),harvested by centrifugation, and washed in RPMI 1640 media (GibcoBRL,Gaithersburg, Md.). 5×10⁴ activated T cells/well (PHA-blasts) wereincubated with varying amounts of IL-4 or mutein in RPMI 1640 mediacontaining 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 2 mML-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycinsulphate in 96 well plates for 72 hrs at 37° C., pulsed with 1 μCi³H-thymidine (DuPont NEN®, Boston, Mass.)/well for 6 hrs, harvested, andradioactivity was measured in a TopCount™ scintillation counter (PackardInstrument Co., Meriden, Conn.).

Example 4 HUVEC IL-6 Secretion Assay

Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics® Corp. (San Diego, Calif.), and maintained as per supplier'sprotocols. Cells (passage 3 to 6) were harvested by incubation withTrypsin/EDTA, washed, and plated at subconfluent densities in 48-wellplates in EGM® media (Clonetics® Corp., San Diego, Calif.) containingbovine brain extract (BBE; Clonetics® Corp., San Diego, Calif.). Atconfluency (3-4 days at 37° C.), the media was removed and replaced withEGM® media without BBE. 24 hours later, varying concentrations of IL-4or mutein was added to the cells in fresh EGM® without BBE, and allowedto incubate an additional 24 hrs. Supernatants were harvested and theconcentration of IL-6 was analyzed using a human IL-6 ELISA. Theconditions were identical except for antagonist assays, varyingconcentrations of mutein were added to a constant concentration of 100pM IL-4. Briefly, 96-well Immunolon® 2 plates (Dynatech Laboratories,Inc., Chantilly, Va.) were coated with 5 μg/ml anti-human IL-6 MAbCat#1618-01 (Genzyme Diagnostics, Cambridge, Mass.) overnight at 4° C.Human IL-6 standard (Genzyme Diagnostics, Cambridge, Mass.) or sampleswere titrated in duplicate and incubated with the coated plate; afterwashes, secondary antibody rabbit anti-human IL-6 PAb (CaltagLaboratories, South San Francisco, Calif., Cat#PS-37) at a 1:1000dilution was added. The presence of bound rabbit anti-IL-6 PAb wasdetected using alkaline phosphatase-coupled donkey anti-rabbit Ig PAb(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.,Cat#711-055-152) diluted 1:2000, and developed using pNPP (SigmaChemical Co., St. Louis, Mo., Cat#N2770 or N1891). Absorbance was readat 405 nm using a Vmax™ kinetic microplate reader (Molecular DevicesCorp., Menlo Park, Calif.).

Example 5 Activities of Muteins

Table 1 summarizes the results of the muteins in the two assaysdescribed above. "EC₅₀, pM" is the effective concentration that producesa 50% maximal response measured in the concentration picomoles/liter.Activity is a function of both potency (EC₅₀) and maximal response(R_(max)). Cell-selective muteins exhibited differential activity ofeither a relative reduction in R_(max) and/or a relative reduction inpotency (increase in EC₅₀) in the HUVEC assay vs. the T cell assay."R_(max), % wt" is the maximal response measured relative to wild-typeIL-4. By definition, wild-type IL-4 gives 100% response. All muteinswere active in the T cell proliferation assay. Muteins R121D, R121E,R121P, and R121T/E122F/Y124Q were more potent than wild-type IL-4 inthis assay, although mutein R121T/E122F/Y124Q had a reduced maximalresponse. Muteins Y124Q, Y124R, and Y124A/S125A had 2-3-fold increasedEC₅₀ values than wild-type, as well as a reduced maximal response.However, they appear to retain a significant proportion of IL-4 activityon T cells. Muteins R121E, Y124Q and R121T/E122FIY124Q had no measurableactivity in the HUVEC assay, making them clearly T cell-selective, andthus selective for the IL-4 receptor expressed on T cells(IL-4Rα/IL-2Rγ). These muteins are IL-4 antagonists on endothelial cellsbecause, although they interact normally with IL-4Rα, they do notactivate the complex IL-4Rα/γ-like subunit. The muteins R121P and Y124Rshow activity in the HUVEC assay, but their EC₅₀ values are increasedbetween 50-150-fold, and have reduced maximal responses relative totheir ability to stimulate T cells. Although these two proteins do notappear to be absolutely T cell-selective, they are preferential fortheir activation of the T cell IL-4 receptor over the HUVEC IL-4receptor.

                  TABLE 1                                                         ______________________________________                                        Muteins with preferential activity on T cells vs. endothelial cells                  1° T cell proliferation                                                              HUVEC, IL-6 secretion                                    Mutein   EC.sub.50, pM                                                                           R.sub.max, % wt                                                                         EC.sub.50, pM                                                                         R.sub.max, % wt                          ______________________________________                                        wildtype IL-4                                                                          150       100       20      100                                      R121A    150       100       20      65                                       R121D    100       40        --      0                                        R121E    65        100       --      0                                        R121F    150       100       20      60                                       R121H    150       80        40      70                                       R121I    100       100       40      50                                       R121K    150       100       100     75                                       R121N    150       100       35      50                                       R121P    100       100       650     45                                       R121T    150       100       20      75                                       R121W    150       100       80      35                                       Y124A    150       50        65      50                                       Y124Q    250       15        200     25                                       Y124R    750       30        250     25                                       Y124S    425       15        350     20                                       Y124T    300       15        350     35                                       Y124A/S125A                                                                            600       60        200     30                                       R121T/E122F/                                                                           15        13        --      0                                        Y124Q                                                                         ______________________________________                                    

Example 6 Biological Response of IL-4 Muteins in HUVEC Assays

FIGS. 3A and B are a series of dose-response plots of HUVEC IL-6secretion by the T cell-selective agonists relative to wild-type. IL-4was included as an internal control on each plate used to assay muteinactivity; a representative curve is shown. FIG. 4A is the dose-responsecurve for R121E versus wild-type IL-4. Similarly, FIGS. 4B-F are thedose-response curves for R121P, Y124Q, Y124R, Y124A/S125A, andR121T/E122FN124Q, respectively, versus wild-type IL-4. Activities havebeen normalized relative to the IL-4 control responses. Muteins R121E,Y124Q, and R121T/E122F/Y124Q demonstrate no activity in this assay.Muteins R121P and Y124R, though showing partial agonist activity in thisassay, are relatively less potent to wild-type IL-4 than they are in the1° T cell assay. Thus, despite their activity, they still demonstratepreferential activation of the T cell IL-4 receptor.

Example 7 Biological Response of IL-4 Muteins in 1° T Cell Assays

FIGS. 4A and B show dose-response curves of the T cell-selective agonistmuteins in a representative assay using cells from a normal donor. IL-4was included as an internal control on each plate used to assay muteinactivity; a representative curve is shown. FIG. 6A is the dose-responsecurve for R121E versus wild-type IL-4. Similarly, FIGS. 6B-F are thedose-response curves for R121P, Y124Q, Y124R, Y124A/S125A, andR121T/E122F/YI124Q, respectively, versus wild-type IL-4. Activities havebeen normalized relative to the IL-4 control responses. Muteins R121E,R121P, and R121TIE122F/Y124Q are more potent than wild-type IL-4,although R121T/EI122F/Y124Q is only a partial agonist in this assay.Although not as potent as wild-type IL-4 in this assay, muteins Y124Q,Y124R, and YI24A/S1 25A are still effective partial agonists (Rmaxvalues 60-70% of wild-type). In FIG. 6, activity is relative to the IL-4response seen in the same plate for each mutein. Of particular note aremuteins R121E and Y124Q, which show significant activity in the T cellassay yet no apparent activity in the HUVEC assay. Each of these muteinsis a clear T cell-selective agonist.

Example 8 Antagonism of HUVEC IL-4-Induced IL-6 Secretion by T CellSelective Muteins

T cell-selective IL-4 muteins R121E () and Y124Q (∇), and the IL-4antagonist R121D/Y124D (O) (FIG. 7), were titrated against a constantconcentration of 100 pM IL-4. The IL-4 antagonist R121D/Y124Dantagonizes IL-4 with a K_(I) of ˜1.5 nM under these conditions. HUVECdo not express IL-2Rγ, but do express the IL-4 receptor γ-like subunit.The substituted residues of R121E and Y124Q are in the D-helix of IL-4and thus only affect interactions of IL-4 with IL-2Rγ (functionalinteraction) and the γ-like subunit (no or non-functional interaction),but do not affect the ability of these muteins to bind to IL-4Rα. Thus,the T cell-selective agonists R121E and Y124Q, by virtue oftheir abilityto selectively interact with IL-2Rγ on T cells without promotingactivation of the γ-like receptor subunit on endothelial cells, are ableto compete IL-4-induced responses on these endothelial cells (K_(I)˜0.8-1 nM under these conditions). Such antagonism by T cell-selectiveIL-4 muteins may antagonize the effects of endogenously produced IL-4 onendothelial cells during T cell-directed therapy with said muteins.

Examples 9 Biological Response of IL-4 Mutein R121D

The IL-4 mutein R121D (Arg-121 substituted with Asp) was generated asdescribed and assayed in the 1° T cell and HUVEC assays. Referring toFIGS. 8A and 8B, data is shown for IL-4 (0) and R121D () in both the Tcell (FIG. 8A) and the HUVEC (Fig.8B) assays. In the T cell assay, R121Dexhibited an EC₅₀ of ˜100 pM and an R_(max) value of ˜60% relative towild-type IL-4. It was inactive in the HUVEC assay (EC₅₀ =; R_(max)=O). These results demonstrate that the single substitution of Arg-121of human IL-4 with Asp yields a protein that has selective activity on Tcells, and lacks any apparent activity on endothelial cells. Althoughthe mutein R121D is a partial agonist in the T cell assay, it is morepotent than wild-type IL-4. However, like mutein R121E, R121D exhibitsno activity in the HUVEC assay, demonstrating that it is a clear Tcell-selective agonist.

Example 10 Biological Activity of IL-4 Mutein T13D/R121E

The IL-4 mutein T13D/R121E (Thr-13 substituted with Asp together withArg-121 substituted with Glu) was generated as described and assayed inthe 1° T cell and HUVEC assays. Specifically with reference to FIGS.9A-B, in the T cell assay (Panel A), T13D/R121E (▴) exhibited an EC₅₀ of˜100 pM, about 2-3-fold better than IL-4 (O) or R121E (Δ), and anR_(max) value of 100% relative to IL-4. In HUVEC antagonist assays(Panel B), T13D/R121E (▴) was ˜27-fold more potent an antagonist of IL-4activity than R121E (Δ), exhibiting an IC₅₀ of ˜2.2 nM vs. ˜60 nM,respectively. The substitution of Thr-13 to Asp increases the potency ofthe T13D/R121E relative to R121E (both as an agonist in the T cellassay, and as an antagonist in the HUVEC assay), while the substitutionArg-121 to Glu confers T cell-selective activity to T13D/R121E (fullagonist in the T cell assay, IL-4 antagonist in the HUVEC assay).

Example 11 Evaluation of the IL-4 Selective Agonist in the PathologyModel

Adult male cynomolgus monkeys (Macaca fasicularis, Charles River PrimateImports, Boston, Mass.), weighing approximately 4 to 6 kg are utilizedfor these studies. Animals are housed individually in environmentallycontrolled rooms in open mesh cages and provided food twice daily andwater ad libitum. Each animal is fasted for approximately 12 hr prior tothe first day of study.

For each study animals are anesthetized with an intramuscular injectionof ketamine hydrochloride (Ketaset, 10 mg/kg). The upper back area ofeach animal is sheared and cleaned with a 70% alcohol-betadine solution.IL-4, IL-4 selective agonist or vehicle (0.2% human serum albumin, HSA)is injected intradermally into the backs of animals in a volume of 0.1ml using a 1 ml tuberculin syringe. The injected sites are separated byat least 10 cm and marked with an indelible marker. Tissue biopsysamples are obtained using a 6 mm punch biopsy tool and samples areplaced in OCT and snap frozen in liquid nitrogen. Biopsies are obtainedat 0, 4, 8 and 24 hrs post injection.

The systemic response to IL-4, IL-4 selective agonist or vehicle isassessed in the following manner. Test article is administeredsubcutaneously twice daily (approximately 10-12 hr apart) over fourconsecutive days in a volume of 0.1 ml/kg at dosages of 0, 2.5, 25 or250 ug/kg resulting in a total daily dose of 0, 5, 50 and 500 ug/kg,respectively. A peripheral blood sample is obtained from each animalprior to the first injection of vehicle or IL-4 and at the beginning ofeach day of the study, and aliquots analyzed for complete blood cellcounts and differentials, and flow cytometry analysis of peripheralblood mononuclear cell surface markers. The remainder of the bloodsample is centrifuged and plasma aliquots stored at -70° C. forsubsequent analysis of chemokine levels.

Frozen sections are prepared and allowed to equilibrate to roomtemperature, air dried and fixed in acetone at 4 C for 5 minutes. Slidesare transferred to 10 mM PBS with 0.1% BSA for 5 minutes. VCAM-1 islocalized with the use of C313.3, a monoclonal antibody to humanVCAM- 1. An irrelevant, isotype-matched immunoglobulin at theappropriate concentration is used as a negative control. Endogenousbiotin is blocked using the Vector Biotin blocking kit (VectorLaboratories, Burlingame, Calif.). Sections are incubated 1.5 hr at roomtemperature in a humid chamber with the indicated antisera diluted inPBS with 0.1% BSA and 1% normal rabbit serum. After three washes withPBS, the slides are stained with the Vector ABC Elite kit according tomanufacturers directions and the antibody conjugate detected byincubating the slides in 3-amino-9-ethylcarbazole/hydrogen peroxide (AECsubstrate kit, Vector). Sections are washed thoroughly in O.lM acetatebuffer, washed in distilled water and mounted in Lerner AQUA-MOUNT(Lerner Laboratories, Pittsburgh, Pa.).

Specimens are scored by two independent observers in a blinded mannerusing set scales between 0 and 3+, designed to assess the intensity aswell as distribution of staining. The scoring system for VCAM-Iexpression is: 0 absent or faint staining of occasional vessel; 1+faintstaining of several vessels; 2+moderate intensity staining of mostvessels; 3+intense staining of most vessels. Blood vessels areidentified in serial sections stained for von Willebrands Factor(polyclonal rabbit anti-human VWF; Dakoplatts, Carpinteria, Calif.).

Analysis of erythrocyte count, hematocrit, leukocyte count and plateletcounts are performed on heparinized blood samples with a Serono 9000Blood Analyzer (Baker Diagnostics, Allentown, Pa.). Leukocytedifferentials are evaluated on Diff-Quick stained blood smears where atotal of two hundred cells are counted and the percentage of each celltype was recorded.

Analysis of peripheral blood mononuclear cell (PBMC) surface markers isperformed in the following manner. A 4 ml sample of heparinized blood isdiluted in Hanks Balanced Salt Solution (HBSS, without Mg++or Ca++) andlayered onto 4 ml of Percoll (1.070 gm/ml density). The tubes arecentrifuged at 1800 rpm (Beckman GS-6R) for 20 minutes at 24 C. Thelymphocyte containing layer is aspirated and centrifuged at 1100 rpm for10 minutes. The resultant cell pellet is resuspended in 6 ml ofphosphate buffered saline (PBS) containing 0.1% Azide and 5% goat serum.Aliquots of 1 ml are utilized for cell surface marker analysis asdescribed below.

Antibodies against CD2, CD4, CD8, CD1 lb,.CD16, CD25, CD49 and HLA-DR(R&D Systems, Minneapolis, Minn.) are utilized for analysis by flowcytometry. Twenty ul aliquots of marker antibodies are incubated with 1ml aliquots of cell suspension in the dark for 60 minutes at 4 C, andsamples centrifuged (1000 rpm, 10 min at 4 C). The pellets are washedthree times with 1 ml of PBS containing 0. 1% azide and 5% goat serum,followed by FACs analysis.

Plasma samples obtained during each study are analyzed for levels ofMCP-1 by specific ELISA. Briefly, 96 well plates (Nunc, Kamstrup,Denmark) are coated with 50 ul/well rabbit anti-MCP-l for 16 hr at 4 Cand then washed in PBS, pH 7.5, 0.05% Tween-20 (wash buffer). Nonspecific binding sites are blocked with 2% BSA in PBS (200 ul) and theplates incubated for 90 minutes at 37 C. Plates are rinsed three timeswith wash buffer, and diluted (neat, 1:5 and 1:10) test sample (50 ul)in duplicate is added, followed by incubation for I hr at 37 C. Platesare washed four times, and 50 ul/well biotinylated rabbit anti-MCP-1 isadded for 45 minutes at 37 C. Plates are washed four times,streptavidin-peroxidase conjugate (100 ug/ml) (Dakopatts, Carpinteria,Calif.) is added and the plates are incubated for 30 minutes at 37 C.The plates are washed three times, and 100 ul chromogen substrate (0.67mg/ml orthophenylenediamine dichloride (Dakopatts, Carpinteria, Calif.)is added. The plates are incubated at 25 C for 6 minutes and thereaction is terminated with 50 ul/well of 3 M H₂ SO₄ solution in washbuffer plus 2% FCS. Plates are read at 490 nm in an ELISA reader.Standards are 0.5 log dilutions of recombinant MCP-1 from 100 ng/ml to 1pg/ml (50 ul/well). The ELISA consistently detects MCP-lconcentrations >50 pg/ml.

Example 12 Treatment of Multiple Sclerosis with IL-4 Selective Agonist

The use of an animal model as a predictor for pharmacological utility inhumans is a well-accepted research tool. Initial testing of the IL-4selective agonist for multiple sclerosis (MS) is conducted in a marmosetmodel using recombinant human IL-4 selective agonist protein. Thesestudies are conducted to examine the effect of prophylactic andtherapeutic treatment on disease induction and severity for both theacute symptomology as well as chronic relapsing-remitting disease.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+T cell-mediatedautoimmune, inflammatory disease of the central nervous system.Induction of EAE is induced in marmosets (C jacchus) weighing 300 to 400gm by immunization with 200 mg of fresh-frozen postmortem human brainwhite matter homogenate (BH) emulsified with complete Freund's adjuvant(CFA) containing 3 mg/ml of killed Mycobacterium tuberculosis asdescribed in Massacesi et al., Ann. Neurol., 37:519 (1995). On the dayof immunization and again 2 days later, 10¹⁰ inactivated Bordetellapertussis organisms are diluted in 10 ml of saline solution andadministered intravenously.

EAE is assessed by clinical and pathological criteria. A standardizedscoring system is employed to record the severity of clinical disease:0=normal neurological findings; 1=lethargy, anorexia, weight loss;2=ataxia, and either paraparesis/monoparesis, sensory loss, or brainstemsyndrome including gaze palsy, or blindness; 3=paraplegia or hemiplegia;4=quadriplegia.

Magnetic resonance imaging (MRI) has been shown to be a useful techniqueto characterize early as well as late immune mediated lesions of MS(Stewart et al., Brain, 114:1069 (1991). MRI is used to evaluate animalsafter immunization to monitor progression of disease over time. MRI datais collected on a Picker International NMR Cryogenic `2000` system,operating at a field strength of 0.15 Tesla; a receiver coil with anaperture of 15 cm to obtain the images. Multislice spin-echo andinversion-recovery pulse sequences are employed. Echo-delays times ofeither 40 and 60 ms, or 40 and 80 ms are used in the spin-echosequences. In the inversion-recovery sequences the 180 -90 interpulsedelay is 400 ms.

Marmosets are anesthetized with ketamine hydrochloride and placed in thescanner using a laser available for patient alignment such that theinner canthi of the eyes are aligned perpendicular to the direction ofthe static magnetic field. Animals are scanned before immunization andthen daily from day 9 after immunization. Prior to scanning each day,animals are checked for signs of neurological impairment.

Animals are sacrificed at different times after immunization. The CNS isremoved and fixed in 10% formalin. Paraffin sections of brain and spinalcord are prepared and stained with hematoxylin and eosin. Each coronalbrain section or horizontal spinal cord section is analyzed forhistopathological findings of inflammation and demyelination accordingto an arbitrary scale: inflammation; 0=no inflammation present, +=rareperivascular cuffs/average whole section; ++=moderate numbers ofperivascular cuffs/section; may have meningeal inflammation;+++=widespread perivascular cuffing and parenchymal infiltration byinflammatory cells. Demyelination score; 0=no demyelination present;+=rare foci of demyelination; ++=moderate demyelination; +++=extensivedemyelination with large confluent lesions.

For pretreatment studies on acute disease pathology, test drug isadministered subcutaneously at a dosage range between 1 and 500 ug/kgfollowing a dosing regimen of 1 administration per day to Iadministration per week prior to the onset of disease symptoms. Fortherapeutic intervention in existing disease, test article isadministered subcutaneously at a dose range between 1 and 500 ug/kgfollowing an extended dosing regimen of 1 treatment per day to 1treatment per week over the course of several months.

Example 13 Treatment of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a debilitating inflammatory disease inwhich chronic activation of resident and infiltrating synovial cellscauses destruction of cartilage and bone and leads to fibrosis and lossof function. Cytokines released from activated T cells are thought toplay a role in the maintenance of the chronic inflammatory reaction.

RA is induced in DBA/1 mice using type II collagen as described byJoosten et al., Arthritis & Rheumatism; 39:797 (1996). Collagen inducedarthritis (CIA) is induced by immunizing mice via intradermal injectionat the base of the tail with 100 ul of emulsion containing 100 ug ofcollagen. On day 21, animals are given a intraperitoneal boosterinjection of type II collagen (100 ug) dissolved in phosphate bufferedsaline (PBS).

Assessment of CIA is performed by examining the mice visually for theappearance of arthritis in the peripheral joints and scores forarthritis severity are assigned. Mice are considered to have arthritiswhen significant changes in redness and/or swelling is noted in thedigits or in other parts of a minimum of 2 paws.

Clinical severity of arthritis is scored on a scale of 0-2 for each pawaccording to changes in redness and swelling (0=no change,0.5=significant, 1.0 =moderate, 1.5=marked and 2.0=severe maximalswelling and redness. Scoring is assessed by at least two blindedobservers.

At the end of the study, some of the animals are sacrificed and paw andjoint tissue is obtained for pathological and histopathologyexamination. The tissue is processed for immunohistochemical staining(frozen sections) or fixed and embedded in paraffin, sectioned andstained with H&E for analysis of cellular infiltration.

Evaluation of a murine analog of the IL-4 selective agonist of thepresent invention in the CIA model is performed with the use of a murineequivalent protein molecule. One of ordinary skill in the art is capableof comparing the murine IL-4 structure with the human IL-4 structure,generating parallel murine IL-4 muteins, and making any necessaryadjustments based on responses in in vitro assays utilizing cell linesexpressing either IL-4Rα/IL-2Rγ or IL-4Rα/γ-like subunit in a manneranalogous to that used for human IL-4 muteins with T cells and HUVEC.Animals are dosed one day prior to the booster administration ofcollagen and kept on a dosing regimen ranging between once a day to oncea week for the duration of the study (40+days). Animals are dosed with arange of concentrations of IL-4 selective agonist ranging between 1 to100 ug/kg.

Example 14 Treatment of Insulin Dependent Diabetes Mellitus (IDDM)

There is some evidence in the literature of Th1 cell involvement in IDDMin humans and animal models of human disease. Nonobese diabetic (NOD)mice are utilized to examine the efficacy of a murine IL-4 equivalent ofIL-4 selective agonist in treating IDDM. One of ordinary skill in theart is capable of comparing the murine IL-4 structure with the humanIL-4 structure, generating parallel murine IL-4 muteins, and making anynecessary adjustments based on responses in in vitro assays utilizingcell lines expressing either IL-4Rα/IL-2R|65 or IL-4Rα/γ-like subunit ina manner analogous to that used for human IL-4 muteins with T cells andHUVEC. Prediabetic NOD mice (approximately 7 wks) exhibit aproliferative unresponsiveness in vitro after T cell stimulation. Thetiming of tmis unresponsiveness is not related to insulitis and persistsuntil the onset of diabetes which occurs at 24 wks of age.

Evaluation of the IL-4 selective agonist in NOD mice is conductedsimilar to studies reported by Rapoport et al., J. Exp Med; 178; p. 87(1993). NOD mice are injected with test material at approximately 3 wksof age following a dosing regimen of once daily treatment or once a weektreatment over the course of 12 weeks until the mice are 15 wks old. Acontrol group of animals will receive treatment with a inert proteinequivalent.

Mice will we tested for glycosuria using Tes-Tape and diagnosed fordiabetes as determined by being glycosuria for at least two consecutiveweeks. At the end of 52 wks, animals are sacrificed to obtain variousorgans and tissue for pathology evaluation. Tissue from the pancreas,submandibular salivary glands and kidney from each mouse is fixed andembedded in paraffin, sectioned and stained. Aldehyde fuchsin stainingof pancreas sections is used to examine the extent to which insuliticinfiltrates have reduced the mass of granulated β cells. Splenicleukocytes are counted by FACScan analyses using anti-Thγ-1.2, anti-CD4and anti-CD8 mabs in ascites as described by Zipris et al., J. Immunol146; p. 3763 (1991).

Other embodiments of the invention will become apparent to one of skillin the art. This invention teaches how to obtain muteins notspecifically described herein but which have T cell activating abilityand reduced endothelial cell activating ability, and thereby thosemuteins come within the spirit and scope of the invention. The conceptand experimental approach described herein should be applicable to othercytokines utilizing heterologous multimeric receptor systems, inparticular IL-2 and related cytokines (e.g., IL-7, IL-9 and IL-1 5),IL-1 0, interferon α, and interferon γ.

SEQUENCES

The following sequences are contained within this application:

SEQ ID NO: 1: hIL-4 (amino acid)

SEQ ID NO: 2: hIL-4 (amino acid, cDNA)

SEQ ID NO: 3: R121A (amino acid, cDNA)

SEQ ID NO: 4: R121D (amino acid, CDNA)

SEQ ID NO: 5: R121E (amino acid, cDNA)

SEQ ID NO: 6: R121F (amino acid, cDNA)

SEQ ID NO: 7: R121H (amino acid, cDNA)

SEQ ID NO: 8: R1211(amino acid, cDNA)

SEQ ID NO: 9: R121K (amino acid, cDNA)

SEQ ID NO: 10: R121N (amino acid, DNA)

SEQ ID NO: 11: R121P (amino acid, cDNA)

SEQ ID NO: 12: R121T (amino acid, cDNA)

SEQ ID NO: 13: R121W (amino acid, CDNA)

SEQ ID NO: 14: Y124A (amino acid, CDNA)

SEQ ID NO: 15: Y124Q (amino acid, cDNA)

SEQ ID NO: 16: Y124R (amino acid, cDNA)

SEQ ID NO: 17: Y121S (amino acid, CDNA)

SEQ ID NO: 18: R121T (amino acid, cDNA)

SEQ ID NO: 19: Y124A/S125A (amino acid, cDNA)

SEQ ID NO: 20: T13D/R121E (amino acid, cDNA)

SEQ ID NO: 21: R121T/E122FNI124Q (amino acid, cDNA)

SEQ ID NO: 22: 5'PCR Primer, IL-4

SEQ ID NO: 23: 3'PCR Primer, IL-4

SEQ ID NO: 24: Mutagenesis Primer for R121A

SEQ ID NO: 25: Mutagenesis Primer for RI21D

SEQ ID NO: 26: Mutagenesis Primer for R121E

SEQ ID NO: 27: Mutagenesis Primer for R121F

SEQ ID NO: 28: Mutagenesis Primer for R121H

SEQ ID NO: 29: Mutagenesis Primer for R121I

SEQ ID NO: 30: Mutagenesis Primer for R21K

SEQ ID NO: 31: Mutagenesis Primer for R121I

SEQ ID NO: 32: Mutagenesis Primer for R21P

SEQ ID NO: 33: Mutagenesis Primer for R121T

SEQ ID NO: 34: Mutagenesis Primer for R121W

SEQ ID NO: 35: Mutagenesis Primer for Y124A

SEQ ID NO: 36: Mutagenesis Primer for Y124Q

SEQ ID NO: 37: Mutagenesis Primer for Y124R

SEQ ID NO: 38: Mutagenesis Primer for Y124S

SEQ ID NO: 39: Mutagenesis Primer for Y124T

SEQ ID NO: 40: Mutagenesis Primer for Y324A/S125A

SEQ ID NO: 41: Mutagenesis Primer for T13D

SEQ ID NO: 42: Mutagenesis Primer for R12IT/EI22F/YI24Q

Note: for the T13D/R121E mutein, the primers SEQ ID NOs: 26 and 41 areused.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 42                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 129                                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #Interleukin-4 proteinION: human                                              -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - His Lys Cys Asp Ile Thr Leu Gln Glu Ile Il - #e Lys Thr Leu Asn             #                15                                                           - Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Gl - #u Leu Thr Val Thr             #                30                                                           - Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr Gl - #u Lys Glu Thr Phe             #                45                                                           - Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Ty - #r Ser His His Glu             #                60                                                           - Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gl - #n Gln Phe His Arg             #                75                                                           - His Lys Gln Leu Ile Arg Phe Leu Lys Arg Le - #u Asp Arg Asn Leu             #                90                                                           - Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Va - #l Lys Glu Ala Asn             #                105                                                          - Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Le - #u Lys Thr Ile Met             #               120                                                           - Arg Glu Lys Tyr Ser Lys Cys Ser Ser                                                         125                                                           - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #IL-4 protein DESCRIPTION: human                                              -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1A                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG GCT GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Ala Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1D                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG GAC GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Asp Gl - #u Lys Tyr Ser Lys               #               1505                                                          #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1E                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG GAA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Glu Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1F                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG TTT GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Phe Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1H                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG CAC GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met His Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1I                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG ATA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Ile Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1K                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AAA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Lys Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1N                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AAC GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Asn Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1P                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG CCA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Pro Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1T                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG ACT GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Thr Gl - #u Lys Tyr Ser Lys               #               1505                                                          #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1W                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG TGG GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Trp Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/Y12 - #4A                                    -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC GAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG GCA GA - #G AAA GCA TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Ala Gl - #u Lys Ala Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/Y124 - #Q                                     -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA CAA TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Gln Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/Y124 - #R                                     -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA CGA TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Arg Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/Y124 - #S                                     -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA TCA TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Ser Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/Y124 - #T                                     -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA ACA TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Thr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/Y124 - #A/S125A                               -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GA - #G AAA GCT GCT AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Gl - #u Lys Ala Ala Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: IL-4/T13D - #/R121E                                -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA GAT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Asp Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG GAA GA - #G AAA TAT TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Glu Gl - #u Lys Tyr Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 462                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: hIL-4/R12 - #1T/E122F/Y124Q                        -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CT - #G TTC TTC CTG CTA             #45                                                                           Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu               #                15                                                           - GCA TGT GCC GGC AAC TTT GTC CAC GGA CAC AA - #G TGC GAT ATC ACC             #90                                                                           Ala Cys Ala Gly Asn Phe Val His Gly His Ly - #s Cys Asp Ile Thr               #                30                                                           - TTA CAG GAG ATC ATC AAA ACT TTG AAC AGC CT - #C ACA GAG CAG AAG              13 - #5                                                                      Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Le - #u Thr Glu Gln Lys               #                45                                                           - ACT CTG TGC ACC GAG TTG ACC GTA ACA GAC AT - #C TTT GCT GCC TCC              18 - #0                                                                      Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Il - #e Phe Ala Ala Ser               #                60                                                           - AAG AAC ACA ACT GAG AAG GAA ACC TTC TGC AG - #G GCT GCG ACT GTG              22 - #5                                                                      Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Ar - #g Ala Ala Thr Val               #                75                                                           - CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GA - #C ACT CGC TGC CTG              27 - #0                                                                      Leu Arg Gln Phe Tyr Ser His His Glu Lys As - #p Thr Arg Cys Leu               #                90                                                           - GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AA - #G CAG CTG ATC CGA              31 - #5                                                                      Gly Ala Thr Ala Gln Gln Phe His Arg His Ly - #s Gln Leu Ile Arg               #                105                                                          - TTC CTG AAA CGG CTC GAC AGG AAC CTC TGG GG - #C CTG GCG GGC TTG              36 - #0                                                                      Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gl - #y Leu Ala Gly Leu               #               120                                                           - AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AG - #T ACG TTG GAA AAC              40 - #5                                                                      Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Se - #r Thr Leu Glu Asn               #               135                                                           - TTC TTG GAA AGG CTA AAG ACG ATC ATG ACC TT - #C AAA CAG TCA AAG              45 - #0                                                                      Phe Leu Glu Arg Leu Lys Thr Ile Met Thr Ph - #e Lys Gln Ser Lys               #               150                                                           #      462                                                                    Cys Ser Ser End                                                                       153                                                                   - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #PCR Primer, IL-4CRIPTION: 5'                                                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24TCAC CTCC                                                  - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 29                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #PCR Primer, IL-4CRIPTION: 3'                                                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #            29    GAAC ACTTTGAAT                                             - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121A                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CTGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121D                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #           30     GACG AGAAATATTC                                            - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121E                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #           30     GAAG AGAAATATTC                                            - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121F                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #27:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   TTGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121H                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #28:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   ACGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121I                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #29:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   TAGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 30:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121K                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #30:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   AAGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 31:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121N                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #31:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   ACGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 32:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121P                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #32:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #           30     CCAG AGAAATATTC                                            - (2) INFORMATION FOR SEQ ID NO: 33:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121T                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #33:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CTGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 34:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121W                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #34:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   GGGA GAAATATT                                              - (2) INFORMATION FOR SEQ ID NO: 35:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124A                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #35:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CATC AAAGTGTT                                              - (2) INFORMATION FOR SEQ ID NO: 36:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124Q                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #36:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   AATC AAAGTGTT                                              - (2) INFORMATION FOR SEQ ID NO: 37:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124R                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #37:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   GATC AAAGTGTT                                              - (2) INFORMATION FOR SEQ ID NO: 38:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124S                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #38:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CATC AAAGTGTT                                              - (2) INFORMATION FOR SEQ ID NO: 39:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124T                 -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #39:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CATC AAAGTGTT                                              - (2) INFORMATION FOR SEQ ID NO: 40:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 33                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/Y124A/S125A           -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #40:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #         33       AGCT GCTAAGTGTT CGA                                        - (2) INFORMATION FOR SEQ ID NO: 41:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 28                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/T13D: T13D                           substitution                                                   -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #41:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   ATTT GAACAGCC                                              - (2) INFORMATION FOR SEQ ID NO: 42:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 34                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Mutagenes - #is Primer, IL-4/R121T/E122F/Y124Q     -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #42:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #        34        ACCT TCAAACAGTC AAAG                                       __________________________________________________________________________

We claim:
 1. A polypeptide comprising a human IL-4 mutein numbered in accordance with wild-type IL4 comprising individual substitutions at position 121, wherein said individual substitutions are selected from the group consisting ofR to A, F, H, I, K, N, P, T or W,wherein said substitutions substantially preserve native T-cell activating ability but substantially reduce endothelial cell activating ability on the resulting MA mutein, relative to wild-type.
 2. The human IL-4 mutein of claim 1 wherein position 121 is substituted with alanine.
 3. The human IL-4 mutein of claim 1 wherein position 121 is substituted with phenylalanine.
 4. The human IL-4 mutein of claim 1 wherein position 121 is substituted with histidine.
 5. The human IL-4 mutein of claim 1 wherein position 121 is substituted wit isoleucine.
 6. The human IL-4 mutein of claim 1 wherein position 121 is substituted with lysine.
 7. The human IL-4 mutein of claim 1 wherein position 121 is substituted with asparagine.
 8. The human IL4 mutein of claim 1 wherein position 121 is substituted with proline.
 9. The human IL-4 mutein of claim 1 wherein position 121 is substituted with threonine.
 10. The human IL-4 mutein of claim 1 wherein position 121 is substituted with tryptophane.
 11. A pharmaceutical composition comprising an effective amount of a polypeptide comprising a human IL-4 mutein numbered in accordance with wild-type L4 comprising individual substitutions at position 121, wherein said individual substitution(s) are selected from the group consisting ofR to A, F, H, I, K, N, P, T or W,wherein said substitutions substantially preserve native T cell activating ability but substantially reduce endothelial cell activating ability on the resulting IL-4 mutein, relative to wild-type in combination with a pharmaceutically acceptable carrier. 